LETlTERS TO THE EDITOR 2629
Fecal
Lactoferrin
as aMarker of Fecal
Leukocytes
Weread with interest the report of Guerrant et al. (1). It was stated that the fecal lactoferrin assay may beauseful diagnos-tic tool in the evaluation of patients with acute diarrhea
syndrome (ADS), offering some advantages over microscopic
examination for fecal leukocytes. Wewould like to comment on this and report data from a study carried out in Chile in which fecal lactoferrin was measured as described elsewhere
(1) by using anti-human lactoferrin antibody (L-232; Sigma). Titers above 1:60wereconsidered positive.
In agreement with previous reports (1,2) showing that lactoferrin is present in human milk (1 g/liter), colostrum
(7g/liter), and cow milk (0.1 g/liter), we found lactoferrin-positive reactions at dilutions of up to 1:105 in milk samples from humans and cows and pasteurized liquid and infant formula(NANandNIDO;Nestle).Theantibodywasshownto cross-react with bovine lactoferrin,but onlywith human milk did itconsistentlyshowa more coarsepatternofagglutination.
The fecal lactoferrin assaywas evaluated with 63 pediatric patientswith ADS and 25 healthyinfants. Stoolsampleswere
collected for fecal lactoferrin,fecalleukocytes, and microbio-logical analysis (bacterial andparasitic entericpathogensand
rotavirus). The type of milk received 48 h prior to sample collectionwasalso recorded.
Among the 25 control infants, all were negative for fecal
leukocytes, 8 breast-fed infants were positive for fecal lacto-ferrin(5of themattitersover 1:540), and 17infants fed other kinds of milkwerenegative forfecal lactoferrin. The presence of lactoferrin in the stools appeared to correlate
strictly
withbreast-feeding. Since 10 of 63 patients with ADS had been
breast-fed, they were excluded from further analysis. Of 53
patientswith ADS, 28
patients
werepositive
for fecallacto-ferrin, 15 of themwere also
positive
forfecalleukocytes,
and 22 produced samples that hadidentifiable entericpathogens
(enteropathogenic Escherichia coli[EPEC], 11; rotavirus, 5;
Cryptosporidium
spp.,3;
Campylobacter jejuni, 1;
Entamoebahistolytica,
1;andShigella flexneri,
1).
Twenty-five patients
withADS were
negative
for fecallactoferrin,
23 of them werenegative for fecal
leukocytes
andonly
6 had an identifiable pathogen(EPEC, 2;
rotavirus, 3;
andShigella
sonnei,
1).
These results suggest that fecallactoferrincould be a more
sensitive test than fecal
leukocytes
for the evaluation ofpatientswithADS,butoneshould
keep
inmind thepossibility
offalse
positives
withbreast-fedinfants,
evenwhenhigh
titers offecal lactoferrinare detected.REFERENCES
1. Guerrant,R. L.,V.Araujo, E. Soares,K.Kotloff,A. A. M.Lima, W. H. Cooper,and A. G. Lee. 1992. Measurementoffecal lacto-ferrinasamarker of fecalleukocytes.J.Clin.Microbiol.30:1238-1242. 2. Sanchez,L., M.Calvo, andJ. H.Brock. 1992.
Biological
role oflactoferrin. Arch. Dis. Child.67:657-661.
TeresaQuiroga PatriciaGarcia ManuelaGoycoolea
Marcela Potin LuisRodriguez
Pablo Vial
UDAClinicalLaboratoriesand
Department
of
Pediatrics SchoolofMedicinePontificia Universidad Cat6lica de Chile
Santiago, Chile
Author's
Reply
We
greatly appreciate
thethoughtful
letter and additional informationby Quiroga
et al.regarding
our report(4).
Infurther support of their
findings suggesting
that fecallacto-ferrin could bea more sensitivetestthan fecal
leukocytes
forthe evaluation of
patients
with acute diarrhea are severalrecent additional reports from at least six different medical
centers
(1, 2,
6-9).
Specifically, Scerpella
etal.found that 94% oftravelerswith invasivepathogens
had fecallactoferrin,
whileonly
69% hadfecalleukocytes (7).
Inaseparate report,Yong
etal. noted that the fecal lactoferrin assaywas moresensitive(75%)
thanmethylene
bluemicroscopy
(40%)
for the detec-tion ofleukocytes
in Clostridiumdifficile
toxin-positive
fecalspecimens
(9).
Milleret al. notedthat,
in contrast tohealthy
volunteersand
patients
with classicalcholeradiarrhea, patients
with
shigellosis
orenteropathogenic
E.coli,
like those withhigh-titer
C.difficile
toxin,
typically
have a moderate if nothighly
inflammatory
process demonstrableby
elevated fecallactoferrintiters
(6). Finally,
Croft etal.,
Thorntonetal.,
andAndersonet al. all noted thatlactoferrinis 25to 114%better than
methylene
blue fordetecting
invasivepathogens
(1,
2,
8).
Furthermore,
Thornton et al. noted that 33% of 21 stoolspecimens
positive
forlactoferrin
butnegative by
culture hadShigella species
detectedby
PCR(8).
While fecal lactoferrin is
highly
sensitive for an inflamma-tory process,however,
the presence of fecal lactoferrin isby
no meansspecific.
Notonly
are many processescapable
ofeliciting
aninflammatory
enteritis(including idiopathic
inflam-matoryboweldisease),
butQuiroga
et al. raise thepotential
problem
of cross-reactions of certain anti-lactoferrin antibod-ies with human milk lactoferrin in the stools of breast-fed infants.Fortunately,
while the lactoferrin found inproducts
from otherspecies,
such as cow'smilk,
may show somecross-reactionwithcertain antibodies suchasthe L-232
Sigma
antibody
usedby Quiroga
etal.,
this is distinct from thereaction seen with human
lactoferrin.
Although
wecertainly
share
Quiroga's
concern about thispossible
cause of"false-positive"
fecal lactoferrindeterminations,
most diarrheal ill-nesses occurinchildrenafterthey
areweaned(5),
andour ownexperiences
with the anti-human lactoferrinantibody
L-3262 fromSigma Company
and with the Leuko-testasprepared by
TechLab are different from theirs. We did not see
agglutina-tion whenwetested bovine milk. None of five human breast milk
samples
we tested gave lactoferrin latexagglutination
titersof>1:8;
threeshowedagglutination
with the Leuko-testbeads
only
whenundilutedandwerenegative
ata1:2dilutioninbuffer.
However,
stillfurther dilutions showed that these fivemilk
specimens
werepositive
at 1:100 and1:1,000 dilutions,
suggesting
a prozone effect consistent with(albeit
at lowertiters
than)
observationsby
Quiroga
andcolleagues.
Deris-bourg
et al. havereported
thesignificant
difference between theglycans
of humanleukocyte
lactoferrin
and thoseof humanmilk
lactoferrin,
with the humanleukocyte
lactoferrinlacking
twofucoseresidues foundonhumanmilklactoferrin
(although
the authors
reported
noantigenic differences) (3).
In the course of
checking
the Leuko-test kit with frozen human breast milksamples
in ourlaboratory,
we have alsofound an
unexpected
coarseagglutination
with the control normalIgG-coated
beads when tested with human milk spec-imens. Whilethiswasnotseenwith thetestantibody
(which
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2630 LETTERS TO THE EDITOR
clearly positivewith lactoferrincontrols),coarseagglutination with the control antibody as provided by TechLab might providea clue that breast milkmaybepresentin thespecimen. In conclusion, our experience and experiences in several
other laboratorieswouldcertainlyconcurwith that ofQuiroga andcolleagues; fecallactoferrin appearstobea moresensitive test than fecal leukocytes in the evaluation ofpatients with
acute diarrhea. However, as with any laboratory test, this
shouldbeinterpretedin the context of the clinicalpresentation (which, with fecal lactoferrin, may include the possibility of
false positives with breast-fed infantswhen tested with certain
antibodies).
REFERENCES
1. Anderson, D. E., J.Kramp, J.Claridge,M.B.Baker,E. M.Latimer,
and D. M. Lyerly. 1994. Detection and stability oflactoferrin in fecal leukocytes by latex agglutination compared to microscopic
evaluation, abstr.C-398,p. 560.Abstr.94thAnnu. Meet.Am.Soc. Microbiol. 1994.American Society forMicrobiology,Washington,
D.C.
2. Croft,S.,P.Newcomb-Gayman,and K.Carroll.1994.Comparison
oftheLeuko-test latexagglutinationassaywiththemethyleneblue stain for the detection of fecal leukocytes, abstr. C-396, p. 560. Abstr. 94th Annu. Meet. Am. Soc. Microbiol. 1994. American Society forMicrobiology,Washington,D.C.
3. Derisbourg, P., J.-M.Wieruszeski,J.Montreuil,andG.Spilk 1990. Primarystructureofglycans isolated from humanleucocyte lacto-transferrin.Biochem.J.269:821-825.
4. Guerrant, R.L., V.Araujo,E. Soares,K. Kotloff,A.A. M.Lima,
W.H.Cooper,andA. G. Lee. 1992. Measurementoffecal lacto-ferrin as a marker of fecalleukocytes.J.Clin.Microbiol. 30:1238-1242.
5. Guerrant,R. L.,L. V.Kirchhof, D. S.Shields, M. K. Nations,J. Leslie,M.A. de Souza,etal. 1983. Prospective studyof diarrheal illnesses in northeastern Brazil: patterns of disease, nutritional impact,etiologies, and risk factors. J. Infect. Dis. 148:986-997. 6. Miller,J. R.,L.Barrett, K. Kotlof, and R. L. Guerrant.
Antilacto-ferrinantibodylatex beadagglutination assayas arapiddiagnostic
testforinflammatory enteritis. Arch. Intern. Med., in press. 7. Scerpella,E.G., P. C. Okhuysen, J. J. Mathewson, R. L.Guerrant,
E. Latimer, D. Lyerly, et al. 1994. Evaluation of a new latex agglutination test for fecal lactoferrin in travelers' diarrhea. J. Travel Med. 1:4-7.
8. Thornton, S.,T.O'Brien, J. Callahan, T. Sharp, R. Batchelor, P. Rozmajzl,M.Wallace,andJ. Burans. 1994. Evaluation ofarapid latex testfordetectionof lactoferrin instool, abstr. C-397,p.560. Abstr. 94th Annu. Meet. Am. Soc. Microbiol. 1994. American Society forMicrobiology, Washington, D.C.
9. Yong,W.H.,A. R.Mattia,and M.J. Ferraro. 1994. Comparisonof fecal lactoferrin latex agglutination assay and methylene blue microscopy for detection of fecal leukocytes in Clostridiumdi ffcile-associateddisease. J. Clin.Microbiol.32:1360-1361.
Richard L.Guerrant,M.D. ClovisMartins,M.D. Terezinha Silva,M.D.
Divisionof Geographicand International Medicine Charlottesville, Virginia22908
Use of Selective Media
for
Isolating Corynebacterium urealyticum
from
Urine
Specimens
Corynebacteriumurealyticum, formerlyknownas Corynebac-teriumgroupD2,is a newspeciesin thegenusCorynebacterium (3)which has been involvedmainlyinurinarytractinfections but also inendocarditis, pneumonia,peritonitis, osteomyelitis,
and soft-tissue infections (1, 5, 6).The clinical significance of
mostorganismsisolatedfromnormallysterilesitessuchasthe
kidney,the bladder wall, the ureter, and blood is relatively easy
to determine. However,that of organismsisolated from non-sterile areas, suchasurine or sputum, is moredifficulttoassess. Ryan and Murray (4) have recently examined thevalue of selective media for isolation of C. urealyticum from urine samplesas well as determination of the clinical relevance of such isolates.They studied 194 urine samples which had pHs of
.7.0,
findingtwoisolates ofC. urealyticum (prevalence, 1% inurine samples with such a pH) which were not related to the
urinarytractinfectin symptoms. This finding partially confirms those of a previously published paper which included more than9,000 unselected urine samples showing a prevalence of 1.17% with selective media but only 0.038% with nonselective media (7). Unsurprisingly, most or all of the 15 organisms isolated from 13 patients by using selective media were not
involvedin the clinical symptoms, but those isolated from three patients by using nonselective media were (7). De Briel et al.
(2), by studying more than 5,000 unselected urine
samples,
isolated C.urealyticum
in 2.5% (2105 CFU/ml) of the urine samples using selective and nonselective culture media (2). Our rates of C. urealyticum isolation from unselected urine samples are 1.9 and 0.23% with selective and nonselectivemedia,respectively. Again, up to 60% of isolates from
nonse-lectivemedium were clinically significant (5); the rate for those
isolates from selective medium was very low (unpublished data). Selectivemediumforisolating C. urealyticumfromurine sampleshasgreatepidemiological value,but itisnotuseful for
managementof thepatients,as moststrains isolatedonly from selective medium have noclinicalrelevance.
The decision to lookfor C. urealyticum in urine specimens and therefore to extend the incubation of urine cultures is a matter open for discussion. Nevertheless, we do not recom-mend the use of selective media for routine purposes. We
recommend cystine lactose electrolyte-deficient and blood
agars instead. Severalcircumstances, such as kind of hospital,
local prevalence, data from urine sediment, and above all, clinical information communicated to the microbiologist, as
alsostatedby Ryan and Murray(4), should be considered key factorsbefore onedecides to search for this pathogen.
REFERENCES
1. Coyle, M. B., and B. A. Lipsky. 1990. Coryneform bacteria in infectious diseases:clinical and laboratory aspects. Clin. Microbiol. Rev.3:227-246.
2. De Briel,D., J. C. Langs, G.Rougeron,P.Chabot, and A. Le Faou. 1991. Multiresistant corynebacteria in bacteriuria: a comparative studyof therole of
Corynebacterium
group D2 andCorynebacterium jeikeium. J. Hosp. Infect. 17:35-43.3. Pitcher, D., A. Soto, F. Soriano, and P. Valero Guillen. 1992. Classification ofcoryneform bacteria associated with human uri-nary tractinfection(group D2) asCorynebacteriumurealyticum sp. nov. Int. J.Syst.Bacteriol.42:178-181.
4. Ryan,M., and P. R. Murray. 1994.Prevalence ofCorynebacterium urealyticum in urine specimens collected at a university-affiliated medical center. J. Clin.Microbiol. 32:1395-1396.
J. CLIN. MICROBIOL.
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Vol.30,No. 5 JOURNALOFCLINICALMICROBIOLOGY, May1992,p. 1238-1242
0095-1137/92/051238-05$02.00/0
Measurement
of Fecal
Lactoferrin
as a
Marker of Fecal
Leukocytes
RICHARD L. GUERRANT,l* VALTERARAUJO,1 ELIZABETHSOARES,"2 KARENKOTLOFF,3 ALDO A. M. LIMA,2 WILLIAM H.COOPER,' ANDAMELIA GAIL LEE'
DivisionofGeographic Medicine, Department of Medicine, University of Virginia SchoolofMedicine, Charlottesville, Virginia 229081; Center for VaccineDevelopment, University of Maryland, Baltimore, Maryland212283;
and Clinical Research Unit, FederalUniversity of Ceard, Fortaleza, Ceara, Brazil2
Recieved 12 November 1991/Accepted 31January1992
While diarrheal illnesses are extremely common in communities and hospitals throughout theworld, an
etiologic diagnosismaybeexpensiveand cost-ineffective.Althoughthepresenceof fecalleukocytesarehelpful
inthediagnosisandspecific therapy ofinflammatory diarrheas, this requirespromptmicroscopicexamination offecal specimens (preferably obtained in a cuprather than a swab ordiaper) by a trained observer. We
developed a simple, sensitive test for the detection of leukocytes in fecal specimens using antilactoferrin
antibody. Whereas radial immunodiffusion detected 0.02,ugoflactoferrin (LF) per ,ulor .2,000 leukocytes per,ul, latex agglutination (LA) readily detected .0.001 ,igof LFper,ulor .200leukocytesper,ul addedto
stoolspecimens. Despitethe destructionorloss ofmorphologic leukocytesonstorage for1to7daysat4°Cor
placementofspecimenson swabs, measurable LF remained stable. Initial studies of stoolspecimensfrom six
patients with Salmonella orClostridium difficile enteritis were positive and those from three controls were negative forLF by LA.Of 17 children in Brazil withinflammatory diarrhea (.1 leukocyte perhigh-power field), 16 (94%) had LF titers of >1:50 byLA,whereasonly3 of12fecal specimens with <1leukocyte per high-power field on methyleneblue examination and noneof 7 normal control specimens had anLFtiterof
>1:50byLA. Of16 fecalspecimensfrompatientswithC.
difficile
diarrhea (cytotoxin titers, .1:1,000),95%(n = 15)haddetectable LFbyLA(intiters of 1:100to1:800). Finally,of 48 fecalspecimensfromhealthyadult
U.S. volunteers before and after experimental shigellosis and of 29 fecal specimens from children with
documentedshigellosisand hospitalizedcontrols innortheastern Brazil,fecal LF titers rangedfrom 1:200to
>1:5,000in96%(25of26) samplesfrompatientswithshigellosis (and reported positiveforfecalleukocytes), while51 controlsconsistently had fecalLFtitersof<1:200.We concludethatfecal LF isauseful markerfor fecal leukocytes, evenwhen theyare morphologicallyloston swabspecimens orwhenthey aredestroyedon
transportorstorageorby cytotoxicfecal specimens.
Diarrheal illnesses areextremelycommon
throughout
theworld,causing2to 16or moreillnesses per person per year in developed and developing countries (7,
15)
and oftenposing diagnostic and therapeutic questions forphysicians.
The causes of diarrhea include awide variety of etiologic
agents, manyof which have beenrecognized onlyinthe past
two decades (7). However, these agents do notneed tobe
exhaustivelysoughtin everyinstance of thiscommon
prob-lem (6, 23), and the cost of indiscriminate use of etiologic
studies for diagnosis isprohibitive. The costfor each
posi-tiveroutine stool culture result has exceeded
$900
to$1,000
(8, 11).An important diagnosticclue inconsideringwhether diar-rheaisanoninflammatoryor aninflammatoryprocessis the examination for fecal leukocytes (4, 9, 12). If fecal leuko-cytes are present, they suggest an inflammatory process caused by Salmonella species, Shigella species,
Campylo-bacterjejuni,
orClostndiumdifficile.
Although the majority of cases are noninflammatory (rotaviruses, Norwalk-likeviruses, enterotoxigenic
Eschenichia
coli) and often respondtosimpleoral rehydration therapy, it is important to distin-guish theinvasive,inflammatorydiarrheal illnesses that may be more severe and that should be the focus of more extensivediagnostic studies and cultures and/or antimicro-bial therapy (8). However, the methylene blue examination for fecal leukocytes requires that the physician or a skilled
microscopist promptly examine under a microscope mucus
*Correspondingauthor.
from a fecal specimen in a cup. Thespecimen is stained so
thatleukocytes are clearly distinguishable in the fecal debris. This requires the immediateavailability of a skilled person with a microscope to stain and examine fresh fecal speci-mensin the clinic or emergency areas where the patient is seen. It may also bedifficult to obtain fecal specimens in a
cup(these specimens aresuperior to swabor diaper speci-mens for examination of fecal leukocytes; swab or diaper specimens are only 44% sensitive, whereas cup specimens are95% sensitive for fecalleukocyte detection by methylene blue examination of specimens from patientswith culture-documented shigellosis [12]). Therefore, a method that
re-mains sensitive when swabs are used and that remains sensitive when specimens are transported or stored (over-night) would behelpful and might allow a wider application ofaselective diagnosticand treatment algorithm.
Wedevelopedasimple in vitro test for a leukocyte marker that is highly sensitive to the numbers of fecal leukocytes
typicallyfound ininflammatorydiarrheal specimens and that
can be quicklyand easily done either in the clinic or later (after transportation or storage) in the laboratory. Only a minimum amount of training is required to learn how to perform the test.
We initially explored the leukocyte marker leukocyte
esterase (an enzyme used in detecting leukocytes in the urine) (16) and found that, in contrast to its usefulness in testing leukocytes in urine, normal fecal specimens gave a
positive result,so weabandoned further studies with leuko-cyte esterase. Instead we found that lactoferrin, an
iron-binding glycoproteinfound concentrated in secondary
MEASUREMENT OF FECAL LACTOFERRIN 1239
ules in leukocytes (10, 19), was not readily detected in normal stoolspecimens unless neutrophils were added. The neutrophils were then readily detected in fecal specimens when lactoferrin was used as a marker. While we initially demonstrated the feasibility of detecting leukocytes in fecal specimens using a radial immunodiffusion assay for lacto-ferrin, the greater sensitivity and speed of latex agglutination led to a focus on that method, as noted below.
MATERIALSANDMETHODS
Preparation of latex beads. Latex beads(Bacto-Latex 0.81 beads;Difco Laboratories, Detroit, Mich.) were coated with rabbit anti-human lactoferrin (product L-3262; Sigma Chem-ical Company, St. Louis, Mo.) as follows: 2.5 ml of beads wascentrifuged at 1,800 xgfor 30min, washed with 5 ml of glycine buffer (7.3 g of glycine and 10 g of NaCl in 1 liter of distilled water adjusted to pH 8.2 to 8.3), and then
resus-pendedin5 ml ofglycine buffer to provide an approximately
1% suspensionof beads. To this latex bead suspension was added 0.35 ml of undiluted rabbitantilactoferrin antibody, to
providea7% antibody dilution in the bead suspension. The mixture was incubated at 38°C for 1 h, after which the
antibody-coatedbeads were spun and resuspended in5ml of buffertowhich 0.005 g of azide(0.1%) and 0.05 g of bovine serum albumin (1%) were added. The coated bead suspen-sion was then stored at 4°C until use. Twenty microliters of thisantibody-coated latex bead suspension was mixed on a microscope slide with 20 ,ul of sample, andagglutination was graded after 2 min with an unaided eye as follows: 0, no
agglutination; +,barely detectable, trace agglutination with
amilky background; 1+, definite, fine agglutination with a milky background; 2+, definite, fine agglutination with a clearing background; 3+, larger agglutination with a clear
background.
For a negative control, latex beads were prepared as described above, but rabbit anti-human lactoferrin was not added. The assay has been licensed and is being developed
as acommercialproductby Techlab Inc., Blacksburg, Va. (a patentispending).
Isolation of neutrophils. Neutrophilswere obtained from normal heparinized (10 ml) venous blood by a one-step Ficoll-Hypaque separation procedure (Neutrophil Isolation
Medium; Los Alamos Diagnostics, LosAlamos, N.M.)(3). The polymorphonuclear leukocytes (PMNs) were washed three times with Hanks balanced salt solution (HBSS).
Residualerythrocyteswere lysed byhypotonic lysiswith 3 mlof iced 0.22%sodiumchloride solution for 45s andthen with 0.88 ml of 3% sodium chloride solution, this was
followedby theadditionof 5 ml ofHBSSand
centrifugation.
A1:2suspensionof PMNs in diluted stoolwas
prepared
by using normal stool of mixed with 1 ml of HBSS to make a cloudy suspensionandadding
0.5 ml ofpreviouslycounted PMNsto0.5 ml of the stoolsuspension.
A1:2suspensionof PMNs in HBSSwasprepared
as a control forcomparison
with fecalsuspensions.
To standardize our
antibody-coated
latex bead prepara-tion,wedetermined dose-responsecurvesusing
asolution oflactoferrin (L-0520;
Sigma)
diluted in HBSS.Testing of PMNs in fecal specimens after
refrigeration
or placement on a swab. To test for PMNs in specimensthatwere
refrigerated
orplaced
onswabs, aliquots
ofpreviously
countedPMN-stool
suspensions
wererefrigerated
orplaced
on swabs for the indicatedtimes. For swab
specimens,
130 ,ul (we found that this volume saturates theswab)
of apreviously counted PMN-stool
suspension
was usedtosat-3+- 1
2+o
Lactoferrin 1+ Latex
Agglutination
.08 .15 .31 .62 1.25 2.5 5 10
Lactoforrin (ng/pi)
FIG. 1. Sensitivity of the latex agglutination assay for lactofer-rin. Data represent the mean (line) and individual results from four different experiments (0) by using two different preparations of lactoferrin and three different preparations of antibody-coated beads.
urate a rayon-tipped swab (Culturette II collection and transportsystem;MarionScientific,Kansas City, Mo.). The swabwasthenplaced in 390 ,u of HBSS and shaken, and the excess fluid was squeezed out to make a calculated 1:4 dilution that we could examine for leukocytes directly and by thelactoferrin assay.
PMNswerequantified morphologically at time zero and at subsequent intervals by using a ruled Neubauer-type hema-cytometer chamber. To aid in visualizing the nuclei,
meth-ylenebluestainwasincorporatedintothecounting solutions (20 ,u of solution, 20
ptl
ofmethylene blue, 160 ,ul of HBSS).RESULTS
Sensitivity
oflactoferrin latexagglutination assay forlacto-ferrin. Asshownin Fig. 1, the lactoferrin latex agglutination assaydonewith three different antibody-coated bead prep-arations wassensitive to less than 1 ng of purified lactoferrin per,ul, withreadily apparentagglutination of the latex beads. The lowest concentration of
lactoferrin
that consistentlygave 1+ or greater agglutination was 0.31 ng/,u, a concen-tration that, from previously published data (10), would be
expected to be present in 60 PMNs per ,u or60 PMNs per
mm3,
a number much lower than that innormal peripheralblood and substantially lower than that expected in an
inflammatory fecal specimen.
Sensitivityoflactoferrin latexagglutinationassayfor PMNs in HBSS or stool suspensions. By using human PMNs that were separatedbyFicoll-Hypaque andsuspended in HBSS
ornormal stoolsuspensions,thesensitivityof thelactoferrin
latex agglutination assay that gavedetectable trace
aggluti-nationwas60to 140 PMNs perRI,
and instoolspecimens,
120to280 PMNs perRI
gavedefinite 1+to2+agglutination
(Fig. 2), with slightly greater
sensitivity
seen when thePMNs were
suspended
in stoolspecimens
than whenthey
were
suspended
in HBSS. The stoolspecimens,
like thedetergent TritonX-100
(0.1%),
thereforeappeared
torelease lactoferrin. Therewas no further increase in thesensitivity
forPMNs in fecal
suspensions
when 0.1or 1% Triton X-100wasused. This number of PMNswasin the range
expected
from the assay
sensitivity
for lactoferrinand,
again,
wassubstantially
below that which would beexpected
frommicroscopic
examination ofinflammatory
fecalspecimens.
To compare the
feasibility
ofdetecting
leukocytes
by
morphologic
counts orby
latexagglutination
forlactoferrin1240 GUERRANT ET AL.
/- , ,,---...
Lactoferrin 1+,
Latex
Agglutination
|,.x_x
0-
*
~~~~~~~~~HBSS
0.1%Triton0 100 200 300 400 500 600 700 800
Calculated PMN/4I
FIG. 2. Sensitivity of the lactoferrinlatexagglutinationassayfor PMNs in HBSS or stool suspensions. Each line represents a
separate experiment. Stool suspensions(likeTriton X-100) appear to release lactoferrin and, thus, increase the sensitivity of the lactoferrin latexagglutination assay.Thesensitivityof the lactofer-rinlatexagglutinationassaythatgavedefinitefineagglutination(1+) was alactoferrincontentof 120to240PMNsper,ul.
after storage in a refrigerator or on swabs, the number of
PMNs inthesuspensionswerecounted and then thePMNs
wereplacedin1% Triton X-100orinacloudy
suspension
ofnormal stool orboth.Then, we reexamined thesuspension
forPMN counts by
microscopy
and lactoferrin titerby
thelatexagglutination assay
immediately
and after storage in a refrigerator or on swabs for 1 to 6 days (Fig. 3 and 4). In contrast tothe labilityofPMN numbersand morphologies seen after refrigeration, lactoferrin titers were remarkablystableevenwhen thestoolorTriton X-100
suspensions
were refrigerated for severaldays.There was a striking loss of PMN numbers after PMNs
were placed on swab specimens
(Fig. 4).
In contrast, as quantifiedbydeterminingthe lactoferrin titer ofleukocytesin fecal suspensions placed on swab specimens remained
stable. Lactoferrin titerswere
relatively
stable afterPMNs were placed in a fecal suspension and thenplaced
on rayon-tippedswabs(incomparisonwith the lactoferrintitersthatweredetermined beforeplacementof the
suspension
on swabs). Incontrast, the number ofmorphologically
evident leukocyteswasextremelyvariable,
withameanloss of 3to 4 log units afterplacementon aswab for 1to 6days.Clinical studies. Initial
pilot
studies of stool specimensfromsixpatientswithSalmonella
(n
=2)
orC.difficile
(n
=1:1600
1:800
Lactoferrin
Latex 1:400
Agglutination (LFLA titers)
(-) 1:200
1:100
T
It
--I., . T+S S
it It
it
T+S
X b
s ".
., s~~~~~~0
-1 0 1 2 3 4 5
Days
*107
,o6
-10
3
,<10
7
FIG. 3. Stability of lactoferrin latex agglutinationassay(LFLA)
titers after refrigeration of specimens for 1 to 6 days. Each line representsaseparateexperiment. Incontrasttothelabilityof PMN
numbers andmorphologiesseenin paired experiments, lactoferrin
titers wereremarkably stable, evenwhen solutionswere
refriger-atedforseveraldays. T, 1% Triton X-100; S, stoolspecimen.
Log
LFLA Titer
(-)
5 Mean
log
.4 PMN/4I (±range)
33
Stool: PMN Suspension
FIG. 4. Stability of lactoferrin latex agglutinationassay(LFLA)
titersdespite theinstabilityof PMNcountswithswabspecimens in
1 to 6 days. "Alone" indicates PMNcounts andlactoferrin titers determinedin thefecal-PMNsuspension just before their placement ontheswabs. Lactoferrinwasconsistently detected in swab spec-imenswithminimalornoloss, incontrast tothestriking variation with a mean 3- to4-log-unitlossofmorphologicPMNcountsafter placementonswabspecimens. Resultsarethemeansandrangeof three and four different experiments for PMN and lactoferrin
measurements,respectively.
4) enteritis were positive and three control stool specimens
were negative for lactoferrin by latex agglutination, thus
demonstrating the feasibility of using this test to examine patient fecal specimens. In preliminary studies to test whether the lactoferrin latexagglutinationmethod correlated withevidence of fecal leukocytesonmicroscopy of methy-lene blue-stained specimens in a field setting, fecal speci-mensin cupsfrom 29 children withacutediarrheapresenting
to aRehydration Center inFortaleza, Ceara, Brazil, where stool cultures are not routinely available, were promptly
examined. Of 17 children whosespecimenshadone ormore leukocytes per high-power field on microscopic examina-tion,wedeterminedbylatexagglutinationthat 16(94%)had fecal lactoferrin at a titer of greater thanorequalto 1:50. In only 3 of 12 children (25%) whose specimens had less than
oneleukocyte perhigh-powerfieldwaslactoferrin presentat
atiter of greater than orequal to 1:50. Inaddition, noneof the seven normal controlspecimensfromhealthyindividuals without diarrhea in Charlottesville, Va., was positive for
lactoferrinat atiter of greaterthanorequalto1:50. When we
systematically examinedbycellculture assay 16 fecal spec-imens frompatientswith nosocomialdiarrheaatthe
Univer-sityofVirginiaHospital that had C.
difficile
cytotoxintiters greater thanor equal to 1:1,000, wefoundbylatex aggluti-nation that 95% (15 specimens) had lactoferrin titers thatranged from 1:100 to 1:800, even though PMNs were not
seen in 6 of 9 of these specimens that were promptly examined after methylene blue staining. Of 14 fecal
speci-mens with cytotoxin titers of 1:10 to 1:100, 9 (64%) had moderatelactoferrintiters(1:100to1:400), while only 1 of 16
specimens (6%)with nodetectablecytotoxin had a lactofer-rin titer of >1:50(1:100).
Finally, we tested fecal specimens from patients with
culture-documentedshigellosis from the University of Mary-land Center for Vaccine Development and Hospital das ClinicasinFortaleza,Ceara, Brazil (Fig. 5). In contrast to 0 of 34 fecal specimens from healthy control adults in the United States that hadatiter of >1:50 or 0 of 17 hospitalized children without diarrheain Fortaleza with fecal lactoferrin titers of>1:200, in 25 of 26 (96%) fecal specimens from adult
J. CLIN. MICROBIOL.
MEASUREMENT OF FECAL LACTOFERRIN 1241
1:6400
1:3200
1:1600'
1:800 1:400 Lactoterrin 1:200
Latex Agglutination1:100
1:50
1:25 1:12.5
1:6.25
<1:6.25
A
.A AA
A&
AAAAiAAaA
L*L*LA
*
Pro-inmsetn Post (n=34)
Healthy U.S. Vol
a
A
AAA
AaaLAAA
AAAA
A
,.o.p.
.f..o- an*gu**o
(nt14) Contol Ca$"
(n.17) (n-12)
unteers HospitalizedChildren In Brazil
FIG. 5. Fecal lactoferrin titers in controlsandpatientswith both experimentaland naturally occurringacute shigellosis. (A) Lacto-ferrin latex agglutination assay titers in the stools of34 different
healthy adult volunteers before experimentalchallenge in vaccine studies and in 14 individuals who developed acute inflammatory diarrhea after experimentalShigellaflexneri infection. (B) Lacto-ferrin latexagglutinationassaytiters in thestools of 12 children with
acute, culture-documented shigellosis and 17 hospitalized control children withoutdiarrheainFortaleza, Ceara,Brazil.
andpediatric patients with documented shigellosis with fecal leukocytesonexaminationby methylene blue staining, fecal
lactoferrin titerswere 21:200 (rangingto 21:5,000).
DISCUSSION
Results of the studies described here demonstrate the practical feasibility of using latex agglutination for determi-nation of fecal lactoferrin as a simple, semiquantitative
marker for PMNs in fecal specimens. Lactoferrin latex agglutination provided a highly sensitive means to quantify PMNs in fecalspecimens. It detected lactoferrinat<1ng/,ul
and <200 PMNsper ,ul, anumber far less thattheexpected number of PMNs ininflammatory fecal specimens. Although fecal leukocytes have long been recognized as being of
diagnostic value, there is little informationon theexpected number of PMNs ininflammatory fecal specimens. Reported numbers ofleukocytes have ranged from multiple cells on
fiveormorehigh-power fields (18)ortwoor moreleukocytes per high-power field seen in 86 to 91% of patients with
Shigella, C. jejuni, and Salmonella infections (versus 0 to
2% for patients with viral, Giardia, or toxigenic bacterial
diarrhea) (1)to >50leukocytesperhigh-powerfieldreported in fecalspecimensfrom 39to85% ofpatientswithshigellosis (20, 21). Speelmanetal.(20) noted that themeannumber of
leukocytespercubic millimeter determinedby
hemacytom-etercounts was28,700 + 4,300for 33 patientswith
shigel-losis (correlated with a mean of 81.6 ± 6 leukocytes per
high-power field). Therefore, oneleukocyteper high-power field would equate to approximately 350 leukocytes per mm3.Therefore,our1:50 dilutionwouldrepresenta
thresh-oldofapproximately8 to 30leukocytesperhigh-powerfield
(basedon thesensitivityofourtestof 60to 200leukocytes per .ul). Lactoferrinis foundinspecific (secondary) granules
in PMNs and isnotfoundinlymphocytesormonocytes (5,
13, 14). Although lactoferrin is present in several bodily secretions,the amounts range from 4.7to 26
ng/pul
in saliva (22)to3.8to218 pig/mgofproteininvaginalmucus(depend-ingonthe time from the patient's lastmenses
[2])
and aresubstantiallylowerthan the amountsof lactoferrinexpected fromthe number ofleukocytes typicallyseeninpatientswith
recognized inflammatory enteritis. At our 1:50 screening
dilution,wemaydetectaslittleas 15 ng oflactoferrinper
,ul,
or about 3,000 PMNs per
Rl.
In our pilot field studies inBrazil, this correlated with 21 PMNper high-power field.
Although Stoll et al. (21) noted that 99% of 304 stool
specimens from patients infected with Shigellaspecies,96% of262 stoolspecimens frompatients infected withC jejuni,
and 98% of 137 stool specimens from patients with
Ent-amoeba
histolytica
showed >1 leukocyte per high-power field, 20to29% ofpatients with rotaviral, enterotoxigenicE.coli, and cholera (all presumably noninflammatory diarrhea)
had 11 to20leukocytesperhigh-powerfield, suggesting that the thresholdseparating patients with primary inflammatory diarrhea from thosewithnoninflammatory diarrhea maybe higher in areaswheremultiple bacterial and parasitic infec-tions are common. Harris et al. (9) noted >25 PMNs per
high-power
field in68% of patients withShigella
orinvasiveE. coli colitis.
Lactoferrin was stable in fecal specimens, even after transportation, storage, swab, ortoxindestroyed the
leuko-cyte
morphology. Preliminary experience
showed that inpatients
withinflammatory
diarrheas causedby Salmonellaspecies
and C.difficile (cytotoxin positive,
antibiotic-asso-ciated
diarrhea),
lactoferrin wasreadily
detectable by thismethod. Fieldstudiesshowed that for94%ofpatients(16of 17 patients) with fecal specimens with one tofive or more
fecalPMNs per
high-power
fieldonmethylene
bluestaining,
lactoferrin latexagglutination
titerswere >1:50. Accordingtothe
sensitivity
ofourassay, thiscorresponds to3,000to12,000 PMNs per
RI,
a concentration range that would beexpected
to correlate with one or more PMNs perhigh-power field in a blood smear. Furthermore, we found a correlation ofC.
difficile
toxin titers with lactoferrin positiv-ity, despite an apparent lack of morphologically evident PMNs in many of the toxin-containing stools. Finally, in contrast to none of 51 controls (34 healthy U.S. adult volunteers and17childrenhospitalized with diagnosesother thandiarrheainFortaleza)who had fecal lactoferrin titers of>1:200, 96%
(25
of26)
ofspecimens
frompatients
withacuteshigellosis (under
experimentalorfieldconditions)
and fecalleukocytes reported on initial examination by methylene
blue
staining
had fecal lactoferrin titers of21:200.
Theonespecimen reported
oninitial examinationtohaveleukocytes
but low lactoferrin concentrations was
unconfirmed,
be-cause examination of all the frozen
specimens
failed torevealleukocytes,
despite
theirpresence inlarge
numbers in the initial examination ofmostspecimens.
Apossible
expla-nation for this includes afalse-positive reading
of the wet mountfor fecalleukocytes
(false-positive
wet mountread-ings
are well documented when fresh fecalspecimens
arealso fixed for
subsequent
staining
and examinationby
oil-immersion
microscopy by
additional trainedmicroscopists
and for
methylene
bluestaining
andlactoferrin
studies).
Alternatively,
frozen fecalspecimens
may notalways
pre-serve lactoferrinor an inhibitormay
rarely
bepresent. Thecontrolsshow
that,
whereashealthy
adults who reside in theUnited States
consistently
have fecallactoferrin
titers ofc1:50,
childrenwithout diarrheamay havefecallactoferrin
titers of as
high
as1:200,
perhaps
reflecting
a subclinicalinflammatory
responseto a commonentericparasite
suchasAscaris
species,
Tichurisspecies
or hookworm(17)
or to subclinicalmalabsorption
ofmilk,
salivary,
or serum pro-tein. In thissetting,
the fecal lactoferrin test mayhelp
todefine mild subclinical
inflammatory
enteritis,
and thethreshold for
highly
significant inflammatory
enteritis may thus beinterpreted
morecautiously, perhaps
witha1242 GUERRANT ET AL.
line zone oftitersrangingfrom 1:50to 1:200suggestingmild inflammation or protein malabsorption. While breastfed in-fants are much less likely to develop serious diarrhea, we
testedstoolspecimens from four healthy breastfed infants in Charlottesville,Va., in whom lactoferrin titers ranged from <1:50 to 1:100. Additional studies with larger numbers of
culture-documented inflammatory and noninflammatory eti-ologies of diarrhea are needed. However, the lactoferrin latexagglutinationtestcorrelates with evidence of PMNs in fecalspecimensdetermined bymethylenebluestaining,and lactoferrinremainsintactevenwhen theleukocyte
morphol-ogyisdestroyedontransferorstorageorby cytotoxicfecal
specimens. Furthermore, it is ofconsiderable practical im-portance that the lactoferrin assay can detect PMNs in specimensobtainedon swabs that adsorb most
morpholog-ically detectable PMNs.
In conclusion, fecal lactoferrin canreadilybedetectedby
a simple latex agglutination assay and provides a useful
marker for inflammatory diarrhea, even when fecal
leuko-cytes are destroyed by transportation, storage, swab, or
toxins. The ultimate utility of this assay in clinical or field settingsand itspotential applicationtospecimensotherthan
stoolspecimens require prospectiveclinical studies in differ-entfield, hospital, clinical, andlaboratorysettings.
ACKNOWLEDGMENTS
This workwassupportedin partbyaUSAID grant fromDiaTech, PATH,Seattle,Wash.,and in partbyaUSAID/CNPQpostdoctoral
fellowshipfor V.Araujo.
We appreciate the technical assistanceof Leah Barrett and the helpof Yatta Jacob inpreparing the manuscript.
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